PUCRS Code: SOP.LMA.

12
Institute of petroleum and Natural Resources
Revision: 00
Environmental Monitoring Laboratory
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Artificial contamination of water samples with reference strains

1. OBJECTIVES AND APPLICATIONS

Assist in the process of artificial contamination of the water sample for
qualitative and quantitative methods for detection of total coliforms and
Escherichia coli, using certified reference materials (CRM).

2. REFERENCES
DOQ-CGCRE-089. Orientação sobre avaliação de desempenho de métodos
analíticos – Microbiologia. 2017.

IDEXX Laboratories. Colilert. Como usar. Available in:

https://www.idexx.com.br/pt-br/water/water-products-services/colilert/

SILVA, Neusely da; JUNQUEIRA, Valéria C A.; SILVEIRA, Neliane F. de A.;

AL, et. Manual de métodos de análise microbiológica de alimentos e água. 5.
ed - São Paulo: Editora Blucher, 2017. E-book. ISBN 9788521212263.
Available in:
<https://integrada.minhabiblioteca.com.br/#/books/9788521212263/>

3. DEFINITIONS AND ACRONYMS

BSC: Biological Safety Cabinet:
E. coli: Escherichia coli
TSA: Tryptone Soy Agar
CFU: Colony-Forming Unit

4. PROCEDURE
4.1 Recommended materials and equipment
 Sterile water;
 TSA culture medium (Tryptic Soy Agar);
 Vortex shaker;
 70% Alcohol;
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Institute of petroleum and Natural Resources
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Environmental Monitoring Laboratory
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Artificial contamination of water samples with reference strains

 Sterile Plastic Inoculation Loop 1 µL;

 Sterile Cell spreaders or Drigalski spatulas;
 Biological Safety Cabinet (BSC);
 Certified reference strains (microorganisms);
 Disposal for tips;
 Refrigerator;
 Kit Colilert;
 Calibrated 100-1000µL micropipette;
 1000 µL sterile tips;

 0.85% saline solution;

 Sterile glass tubes with screw caps.

4.2 Procedure for prior inoculum preparation

It is recommended previously prepare and label:

1. Sterile glass tubes, with screw caps, containing 9 mL of 0.85% saline

solution for performing serial dilutions (10-1 to 10-9);
2. Additional tubes containing 10 mL of 0.85% saline solution (tube 1) and
3. Petri dishes with TSA agar to perform the plating of the dilutions and the
colony-forming units (CFU) analysis.

Also needed is a recent culture of the certified reference strain, grown for 18
to 24 hours as per IT.LMA.10, section 3.4.2, for subsequent inoculation into
tube 1, following the provided recommendations:

1. In Biological Safety Cabinet (BSC), transfer a 1 µL aliquot of the

reference strain to sterile tube containing 10 mL of 0,85% saline solution
(tube 1);
2. Close the tube and homogenize for about 15 seconds using a vortex
agitator;
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Institute of petroleum and Natural Resources
Revision: 00
Environmental Monitoring Laboratory
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Artificial contamination of water samples with reference strains

3. Incubate the tube 1 in bacteriological incubator at a tempeature of 30°C

to 35 °C for approximately 24 hours, for use following day for serial
dilutions.

4.3 Preparation of the inoculum with certified reference strains

1. Prepare sterile glass tubes for serial dilution containing 9 mL of sterile

0.85% saline solution and Petri dishes with TSA medium;
2. Label each tube and TSA with the corresponding dilution, for example

10-1, 10-2, 10-3, 10-4,10-5, 10-6, 10-7, 10-8;

3. Transfer 1 mL from tube 1 to tube 2 (10 -1), containing 9mL of 0,85%

saline solution, close, and homogenize for about 15 seconds;
4. Repeat step 3 for each test tube, transferring 1 mL from the previous
dilution to the subsequent one (as shown in Figure 1).

Figure 1. Representative diagram for inoculum preparation with certified reference strains,
using the serial dilution technique,
 PUCRS Code: SOP.LMA.12
Institute of petroleum and Natural Resources
Revision: 00
Environmental Monitoring Laboratory
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Artificial contamination of water samples with reference strains

5. After the last diluition, transfer 100 µL of each dilution tube to the
corresponding plates (in technical triplicate), using the surface
planting tecnique (spread plate) (figure 2);
6. Spread the inoculum using a sterile Drigalski loop;

Figure 2. Surface inoculation (spread plate) which involves spreading a bacterial suspension to
achieve growth for colony counting (CFU/mL)

7. Incubate the inoculated TSA on bacteriological incubator for about 24

hours, at 30°C to 35°C;
8. Stock the tubes dilution containing tubesin refrigerator (2-8°C) for use
on the following day, while awaiting colony development.

After the incubation period, visually check the colony growth and count the
number of detected colonies.Calculate CFU according to the following formula.
For recording and performing calculations, you can use PLAN.LMA.02.

1
CFU/mL = n° of colonies X Dilution factor X ( )
Inoculated volume
 PUCRS Code: SOP.LMA.12
Institute of petroleum and Natural Resources
Revision: 00
Environmental Monitoring Laboratory
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Artificial contamination of water samples with reference strains

If the count is performed on TSA inoculated with the undiluted sample

and without duplicates, the number of CFUs is equal to the number of colonies.
If conducted, at least in duplicate, the number of CFUs equal the arithmetic
mean of the count obtained on each of the plates.

4.4 Artificial contamination from the water sample

1. After selecting the TSA plate corresponding to the dilution with couting
between 100 – 300 CFU/mL, calculate the sample contamination according
to the example below, also following the recommendations of SOP.GQ.11 –
Table 8.

2 CFU – 100 mL

X– 1000 mL
X = (2*1000) / 100
X = 20 CFU

In which this:

 2 CFU = Target value close to

themethod’s detection limit;

 100 mL = Final sample volume of

themethod;

 1000 mL = volume of solution prepared to

artificially contaminate the samples.

Note 1: The detection limit of the Colilert test for total coliforms and E.coli is 1
organism/100mL.
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Artificial contamination of water samples with reference strains

Note 2: The volume of solution prepared to artificial contaminate the sample

may vary.

2. Based on the result obtained in the previous example, check the colony
count and choose the dilution that contains the approximate CFU value. For
the following example, the dilution with approximate CFU value is 10-7.

1 mL of diluton 10-7 – 20 CFU

X – 20 CFU
X = 1 mL = 1000 µL

In which this:

 20 CFU = Value responding to the diluition 10-7

 20 CFU = Target value

3. Remove the tube with the chosen dilution from the refrigerator and leave at
room temperature;
4. In BSC, add to the solution prepared to artificially contaminate the samples,
the previously calculated volume (item 2) of the reference strain, according
to the chosen dilution tube, as described below:
 Positive control for total of coliforms and E.coli: E.coli.
 Positive control for total of coliforms and negative to E.coli: Klebsiella
aerogenes.
 Negative control for total od coliforms and E.coli: Staphylococcus
aures.
 Negative control: Only sterilized water without inoculum.

Note: For contamination of the negative control, use two log 10 levels above the
concentration level of the target microorganism (E. coli).

5. Add the contents from the Colilert kit to each bottle containing 100 mL of
water contaminated with microorganisms;
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6. Close the bottle and vigorously homogenize;

7. Incubate according to the recommendations in item 4.5.

4.4.1 Qualitative Test

For qualitative evaluation, it is recommended to use artificially contaminated

water samples. For this, it is recommended to perform the water contamination
with a certified reference microbial culture, according to item 4.4 (steps 1 to 6).
It is important to highlight that the starting water samples have to be known as
negative for the target microorganism. To do this, follow the recommendations
in SOP.LMA.07 (item 4.2).

4.4.2 Quantitative Test

For quantitative assays, it is recommended to use watersamples naturally

contaminated with the target microorganism. If this is not possible, artificially
contaminated samples can be used, following item 4.4 (steps 1 to 6). Distribute
the sample into sterile test tubes, according to SOP.LMA.08 (item 4.2).

4.5 Incubation

Incubate the sample at 34 - 36ºC for a minimum of 24 hours (with the Colilert-18
kit, the incubation period is a minimum of 18 hours). For interpretation and
reporting of data, follow the SOP.LMA.07 (for qualitative assays) and/or
SOP.LMA.08 (for quantitative assays).

5. RECORDS

Record the results in FOR.LMA.15 - Microbiological Testing Record and/or

prepare the report according to FOR.GQ.02.
 PUCRS Code: SOP.LMA.12
Institute of petroleum and Natural Resources
Revision: 00
Environmental Monitoring Laboratory
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Artificial contamination of water samples with reference strains

6. REVISION HISTORY

Date of Change in
Revision preparation/Revision document Preparation/Review Approval

Ana Victoria Dunke

00 29/02/2024 Emission Porto/Francine
Melise dos Santos